Host determinants of alphavirus replication

描述(申请人提供)：甲型病毒是人类和牲畜的病原体，分布在世界各地。将一些物种归类为特定病原体，并结合它们对公共卫生的影响，使了解它们与宿主的相互作用和制定干预措施成为高度优先事项。甲型病毒必须通过嗜血性节肢动物媒介传播，在这种媒介中建立了终生持续感染。这种感染模式意味着一种有效但不完整的免疫反应，在保持宿主健康的同时允许病毒复制继续，从而促进病毒传播。理解关键的病毒-宿主相互作用的一个主要障碍是无法从基因上操纵宿主生物体。黑腹果蝇是一个很好的实验系统，用于阐明许多生理和发育过程背后的遗传、分子和生化机制。这项拟议的研究旨在利用果蝇遗传学的力量来确定最近表征的甲型病毒与两种节肢动物先天性免疫反应途径之间的相互作用。在GAL4-UAS误表达系统的控制下，从果蝇基因组中表达了编码报告基因的甲病毒复制子序列，建立了甲病毒复制分析系统。由此产生的病毒RNA的复制可以通过报告基因的活性观察到。使用这种甲型病毒复制子苍蝇系对宿主途径的突变分析表明，IMD-和JAK-STAT途径具有抗病毒作用。这个系统已经被进一步开发，可以产生具有感染性的颗粒。复制子和病毒结构蛋白在果蝇中的共表达导致复制子的产生，该复制子含有能够单轮感染的颗粒。这些基本工具将与果蝇遗传学结合使用，以(I)确定负责激活IMD-和JAK-STAT通路的病毒成分；(Ii)确定参与病毒诱导的先天性免疫反应的宿主基因；(Iii)表征宿主对病毒传播的要求。这项拟议的研究将果蝇遗传学的力量与比较基因组学和分子病毒学相结合，以促进我们对甲病毒与节肢动物宿主之间相互作用的理解。 与公共卫生的相关性：了解病毒与宿主的相互作用对于了解感染的结果和确定阻断传播的手段至关重要。这项拟议的研究旨在使用一个模型遗传系统来检查宿主对甲型病毒的反应。

DESCRIPTION (provided by applicant): Alphaviruses are pathogens of humans and livestock with worldwide distribution. The classification of some species as select agents in conjunction with their impact on public health makes understanding their interaction with the host and development of interventions a high priority. Alphaviruses are obligatorily transmitted by a hematophagous arthropod vector in which a lifelong persistent infection is established. This pattern of infection implies an effective but incomplete immune response that preserves host fitness while allowing virus replication to continue thus facilitating virus transmission. A major impediment to understanding the critical virus-host interactions has been the inability to genetically manipulate the host organism. Drosophila melanogaster represents an excellent experimental system for elucidating the genetic, molecular, and biochemical mechanisms underlying numerous physiological and developmental processes. The proposed research aims to exploit the power of Drosophila genetics to define the recently characterized interactions between alphaviruses and two arthropod innate immune response pathways. A system for the analysis of alphavirus replication has been established in which an alphavirus replicon sequence encoding a reporter gene is expressed from the genome of Drosophila under the control of the GAL4-UAS misexpression system. The resulting replication of the viral RNA is observable through reporter gene activity. Mutational analyses of host pathways using this alphavirus replicon fly line have demonstrated an antiviral role for the Imd- and JAK-STAT pathways. This system has been further developed to produce infectious particles. Co-expression in Drosophila of the replicon and viral structural proteins leads to the production of replicon containing particles capable of a single round of infection. These basic tools will be used in combination with Drosophila genetics to (i) determine the viral components responsible for activation of Imd- and JAK-STAT pathways; (ii) identify host genes involved in virus-induced innate immune response; (iii) characterize host requirements for virus spread. The proposed research combines the power of Drosophila genetics with comparative genomics and molecular virology in order to advance our understanding of the interaction between alphaviruses and an arthropod host. PUBLIC HEALTH RELEVANCE: Understanding virus-host interactions is essential to understanding the outcome of infection and determining means of interrupting transmission. The proposed research aims to examine the host response to alphaviruses using a model genetic system.